丹参酮IIA在人肝微粒体中的代谢研究

Salvia miltiorrhiza (Danshen) has been widely used in China for the treatment of coronary heart disease, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure. Tanshinone IIa (6,7,8,9-Tetrahydro-1,6,6-trimethylphenanthro[1,2-b]furan-10,11-dione), one of the major active constituents of Salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction, anticancer, antibacterial and antiplatelet aggregation activities. As compared to the extensive research of the pharmacological activities of tanshinone IIa, few studies have dealed with its metabolism and pharmacokinetics. Hydroxylation is an important pathway in the metabolism of tanshinone IIa. However, the metabolites and primary cytochrome P450 (CYP) isozymes responsible for tanshinone IIa hydroxylation remain to be determined in human. Here, we characterized tanshinone IIa hydroxylation by human liver microsomes (HLMs) and nine recombinant human CYP (rCYP) isozymes to identify what kinds of metabolites are present and which human CYP isozymes are involved. One hydroxyl metabolite was detected in reactions catalyzed by HLMs and rCYP2A6, and was identified as tanshinone IIb by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. A kinetic study showed that tanshinone IIa hydroxylation by HLMs and rCYP2A6 followed Michaelis-Menten kinetics. The Km values of HLMs and rCYP2A6 for tanshinone IIa hydroxylation were 5.93 ± 0.44 and 5.22 ± 0.31 μM, respectively. A CYP2A6 selective inhibitor (8-methoxypsoralen) inhibited tanshinone IIa metabolism almost completely, with no metabolite detectable, but other CYP-selective inhibitors exerted no inhibitory effects. Rates of tanshinone IIa hydroxylation correlated well (r > 0.98, P < 0.01) with rates of hydroxylation of the CYP2A6 probe substrate coumarin from 15 individual HLMs. In combination, we demonstrate that tanshinone IIa is metabolized by HLMs to a single metabolite (tanshinone IIb) and the tanshinone IIa hydroxylation is specifically catalyzed by CYP2A6 in HLMs. and that makes tanshinone IIa has potential use as an in vitro probe for CYP2A6 catalytic activities